The formation of blood clots does not only limit bleeding in case of injury (haemostasis) but can occlude important arteries or veins, leading to serious organ damage and death. Thrombosis is thus blood clot formation at the wrong time and place.
Upon damage of a vessel, the coagulation (clotting) system is immediately initiated producing thrombin and blood platelets adhering to matrix proteins, which in turn leads to the aggregation of additional platelets into a growing platelet plug in concert with the conversion of fibrinogen in the blood to the insoluble fibrin.
At each step of the coagulation cycle, a clotting factor zymogen undergoes limited proteolysis and itself becomes an active protease. This clotting-factor enzyme activates the next clotting factor zymogen until thrombin is formed which connects fibrinogen to the insoluble fibrin clot. The blood clotting factors include factor I (fibrinogen), factor II (prothrombin), tissue factor (formerly known as factor III), factor IV (Ca2+), factor V (labile factors), factor VII (proconvertin), factor VIII (antihemophilic globulin, or 11AHG11), factor IX (Christmas factor), factor X (Stuart factor), factor XI (plasma thromboplastin antecedent, or “PTA”), factor XII (Hageman factor), factor XIII (fibrin stabilizing factor), and factors HMWK (high-molecular weight kininogen, or Fitzgerald factor), PREK (prekallikrein, or Fletcher factor), Ka (kallikrein), and PL (phospholipid).
Fibrinogen is a substrate for the enzyme thrombin (factor IIa), a protease that is formed during the coagulation process by the activation of a circulating zymogen, prothrombin (factor II). Prothrombin is converted to the active enzyme thrombin by activated factor X in the presence of activated factor V, Ca 2+ and phospholipid. Two separate pathways, called the “intrinsic” and “extrinsic” systems, lead to the formation of activated factor X. In the intrinsic system, all the protein factors necessary for coagulation are present in the circulating blood. In the extrinsic system, tissue factor, which is not present in the circulating blood, is expressed on damaged endothelium, by activated monocytes, by cells in the arteriosclerotic plaque or by cells outside the vessel wall. Tissue factor then acts as the receptor and essential cofactor for the binding of factor VII, resulting in a bimolecular enzyme (tissue factor VIIa) to initiate the extrinsic pathway of coagulation. This mechanism also activates the intrinsic pathway of coagulation.
As a summary, the coagulation system involves a cascade of complex and regulated biochemical reactions between circulating blood proteins (coagulation factors), blood cells (in particular platelets) and elements of an injured vessel wall. Venous thromboembolic disease (deep vein thrombosis, pulmonary embolism, atrial fibrillation) remains a major health issue, with an incidence of 1 to 3 per 1000 individuals per year and a high early mortality rate (Nordstrom et al. (1992) J Intern Med. 232, 155-160; Rosendaal (1997) Thromb Haemost 78, 1-6).
Current anticoagulant therapies primarily consist of heparin (or low molecular weight heparins) and vitamin K antagonists, which are both unsatisfactory and inconvenient. All treatments carry a significant risk of bleeding (Res. Comm. British Thoracic Soc. (1992) Lancet. 340(8824):873-6), which limits both the dose and duration of treatment and may require regular monitoring (Hylek & Singer (1994) Ann Intern Med. 120, 897-902; Cannegieter et al. (1995) N Engl J Med. 333, 11-17). New drugs are currently being developed, but none appears to match optimal criteria of efficacy, safety and convenience.
Antibodies directed to coagulation factors were recently developed as anticoagulant agents. Antibodies directed against Factor IX, Factor IXa, Factor X, Factor Xa, Factor XI, factor XIa, Factor VIII, Factor VIIIa, Factor V, Factor Va, Factor VII, Factor VIIa, thrombin, the Von Willebrand Factor, Tissue Factor and other elements of the coagulation cycle have already been described.
WO 97/26010 discloses antibodies inhibiting coagulation in what is described as “a self-limited manner”. These antibodies are characterized by the fact that high concentrations of such antibodies prolong coagulation tests such as the APTT only in a limited manner and will not render blood unclottable in contrast to high doses of anticoagulant agents such as heparin. However, a limited increase in APTT does not exclude the risk of bleeding. It has not been shown that these antibodies having a so-called “self-limiting neutralizing activity” can avoid completely neutralizing their target coagulation factor, thereby exposing the patient to high bleeding risks. Indeed, in patients with complete deficiency of coagulation factors such as FVIII or FIX, APTT is also prolonged in only a finite manner. The blood of such patients is also not uncoagulable in contrast to blood treated with high doses heparin. However, such patients with severe FVIII or FIX deficiency suffer from dramatic hemorrhagic diseases called hemophilia A or B. As antibodies inhibiting coagulation factors in a “self-limited manner” have biological activities mimicking the blood defect in these patients, they may expose the patients to high bleeding risks.
WO 01/04269 discloses a human monoclonal antibody, Krix-1, which only partially inhibits FVIII activity whatever the (molar) excess of antibody over FVIII. This limited inactivation of FVIII was called a “plateau effect”. By comparison with antibodies having “self-limiting neutralizing activity”, antibodies such as Krix-1 have the advantage that they cannot completely inactivate the target coagulation factor. WO 01/04269 A1 discloses that despite this limited FVIII inactivation, Krix-1 was efficient in preventing thrombosis in a hamster model of venous thrombosis. This antibody was also effective in a mouse model of vena cava thrombosis (Singh et al. (2002) Blood 99, 3235-3240). Krix-1 inhibits about 90% FVIII activity (range 85-95%) in normal human plasma.
Factor FVIII therefore appears as a potential target for anticoagulant drugs. However, it is likely that the bleeding tendency associated to the use of anti-FVIII antibodies will be related to the degree of inhibition of the target coagulation factor. It is therefore important to establish methods to generate antibody preparations with an optimal ratio between efficacy (antithrombotic action) and safety (low bleeding tendency).
So far, all of the anticoagulant agents tested in clinical studies are associated with an important risk of bleeding. Besides, LMWH requires frequent subcutaneous administrations and coumarin derivatives require regular monitoring.
Safer and more efficient methods for the prevention and treatment of venous thromboembolic diseases are therefore desirable. Ideal anticoagulant agents should not carry a risk of bleeding complications or of overdosing. They should not require regular monitoring, be easy to administer and well-tolerated. Finally, an antidote should be available.
As a summary, there is still a stringent need for good anti-coagulant therapies with better safety/efficacy ratios.